Angiogenesis is a fundamental process by means of which new blood vessels are formed. This process is essential in many normal physiological phenomena such as reproduction, development and cicatrization. Angiogenesis is under strict control in these normal biological phenomena; i.e., it is triggered during a brief period of several days and then completely inhibited. However, many pathologies are linked to invasive, uncontrolled angiogenesis. Arthritis, for example, is a pathology caused by damage caused to cartilage by invasive neovessels. In diabetic retinopathy, the invasion of the retina by neovessels results in the patients going blind; neovascularization of the ocular apparatus is the major cause of blindness and this neovascularization dominates at least twenty diseases of the eye. Lastly, the growth and metastasis of many tumors are directly dependent on angiogenesis. The tumor stimulates the growth of the neovessels for its own use. Furthermore, these neovessels present escape routes by means of which the tumors can reach the blood circulatory system and cause metastases in remote sites such as the liver, lungs or bones.
Angiogenesis can present an important therapeutic basis in other pathologies such as cardiovascular diseases, diseases of the peripheral arteries and vascular or cerebral lesions. In fact, the promotion of angiogenesis in damaged areas can lead to the formation of lateral blood neovessels as alternatives to the damaged vessels, thereby providing the damaged area with oxygen and other nutritive and biological factors necessary for the survival of the tissues in question.
The formation of neovessels by endothelial cells involves the migration, growth and differentiation of endothelial cells. Regulation of these biological phenomena is directly linked to genetic expression. An increasing number of studies have shown that the regulation of angiogenesis is implemented via an equilibrium among the factors acting directly on the endothelial cells. These factors can be stimulatory on the one hand, such as (among others) VEGF, FGFs, IL-8, HGF/SF and PDGF. The factors can also be inhibitory, such as (among others) IL-10, IL-12, gro-α and gro-β, platelet factor 4, angiostatin, the human chondrocyte derivative inhibitor, thrombospondin and the leukemia inhibitor. (Jensen, 1998 Surg. Neural., 49, 189-195; Tamatani et al., 1999, Carcinogenesis, 20, 957-962; Tanaka et al., 1998, Cancer Res., 58, 3362-3369; Ghe et al., 1997, Cancer Res., 57, 3733-3740; Kawahara et al., 1998, Hepatology, 28, 1512-1517; Chandhuni et al., 1997, Cancer Res., 57, 1814-1819; Jendraschak and Sage, 1996, Semin. Cancer Biol., 7, 139-146; Majewski et al., 1996, J. Invest. Dermatol., 106, 1114-1119.)
The regulation of angiogenesis as described at present is implemented via an equilibrium of two types of factors:                the angiogenic factors (extracellular polypeptides, primarily mitogenic) acting directly on endothelial cells inducing angiogenesis; and        the angiostatic factors (extracellular polypeptides, also mostly mitogenic and acting on mitogenesis), also acting directly on endothelial cells so as to inhibit angiogenesis.        
The equilibrium between these two types of extracellular factors regulates angiogenesis. It should be noted at this stage that the control of angiogenesis is implemented via the production of angiogenic and angiostatic factors. For example, it has already been shown that the stimulation of the endothelial cell by an angiogenic factor induces the expression of 1) urokinase plasminogen activator (uPA) and its inhibitor PAI-I (Pepper et al., 1990, J. Cell Biol. 111(2), 743-44; Pepper et al., 1996, Enzyme Protein, 49 (1-3), 138-62); 2) matrix metalloproteinases (MMPs) and physiological inhibitors of the activity of these MMPs (TIMPs) (Cornelius et al., 1995, J. Invest. Dermatol., 105(2), 170-6; Jackson and Nguyen, 1997, Int. J. Biochem. Cell Biol., 29(10), 1167-77); 3) inhibitors such as angiopoietin-2 (Ang-2) or thrombospondin-1 (TSP-1) (Mandriota and Pepper, 1998, Circ. Res. 83, 852-859; Oh et al., 1999, J. Biol. Chem. 274(22), 15732-9; Suzuma et al., 1999, American Journal of Pathology, 154, 343-354.) It thus appears that an endothelial cell in the angiogenic state normally produces not only angiogenic factors but also produces angiostatic factors as well. The production of these angiostatic factors enables control of angiogenesis.
Parallel to this operation, endothelial cells stimulated by an angiostatic factor produce not only angiostatic factors, but also produce angiogenic factors for controlling the angiostatic state. This phenomenon has already been described for other types of cells that produce factors implicated in angiogenesis when they are stimulated by an angiostatic factor such as interferon-gamma (Kobayashi et al., 1995, Immunopharmacology, 31(1), 93-101; Arkins et al., 1995, Mol. Endocrinol., 9(3), 350-60; Kodelja et al., 1997, Immunobiology, 197(5), 478-93).
Angiopartnerine is homologous with the negative regulator of the prostaglandin F2 receptor (PTGFRN), (access no. XM—040709, nucleic sequence: 5975 bp, protein sequence: 560 aa), with the protein 6 associated with the human smooth muscle cell (SMAP6) (accession no. AB014734, nucleic sequence: 2197 bp, partial protein sequence: 186 aa), identified by the numbers SEQ ID No. 27 and SEQ ID No. 28 respectively in the attached sequence listing, itself being similar to the regulatory protein of the prostaglandin F2 alpha (FPRP) also designated CD9P-1 and renamed EWI-F by Stipp et al. (2001, J. Biol. Chem., 276, 44, 40545-40554). The sequence GS-N1 comprises the sequences SEQ ID No. 27 and SEQ ID No. 28, presenting 99% of homology with them.
FPRP is a type 1 transmembrane glycoprotein containing 6 extracellular immunoglobulin domains. It was originally identified and characterized by its capacity to combine with prostaglandin F2 alpha and inhibit the binding of this prostaglandin with its receptor. It can also combine with other receptors that are coupled to G protein and contain 7 transmembrane domains, and reduce the ligand-receptor interaction (Orlicky and Nordeen, 1996, Prostaglandins Leukot. Essent. Fatty Acids, 55: 261-268; Orlicky et al., 1998, J. Lip. Res., Vol. 39, 1152-1161). The augmentation of the expression of FPLP has already been associated with cell differentiation, notably that of adipocytes (Orlicky et al., 1998, J. Lip. Res., Vol. 39, 1152-1161). Different studies have shown that CD9P-1 or FPLP is a major partner of two member of the tetraspanin family (also called TM4SF), CD9 and CD81 in protein complexes, combining specifically either with CD81 or with CD9 and CD81 (Charrin S. et al., 2001, J. Biol. Chem.; Stipp et al. (2001, J. Biol. Chem., 276, 7, 4854-4862). The tetraspanins have been implicated in many cellular functions such as adhesion, migration, co-stimulation, transduction of the signal and differentiation, the various functions attributed to the tetraspanins can be linked to their specific combination with the specific partner molecules (Le Naour et al., 2000, Science, 287, 319-321).
The protein CD9 has a broad tissue distribution; it has notably been found in various types of tumors (Si and Hersey, 1993, Int. J. Cancer, 54: 37-43; Miyake et al., 1996, Cancer Res., 56: 1244-1249) as well as in the vessels formed by endothelial cells (Zola et al., 1989, Immunol. Cell Biol.; 67: 63-70). This protein has been implicated in functions such as transduction of the signal, cell adhesion, motility, tumor progression (Ozaki et al., 1995, J. Biol. Chem., 270: 15119-15124; Forsyth, 1991, Immunology, 72: 292-296; Anton et al., 1995, J. Neurosci., 15: 584-595; Shaw et al., 1995, J. Biol. Chem., 270: 24092-24099; Ikeyama et al., 1993, J. Exp. Med., 177: 1231-1237) and notably the adhesion and migration of endothelial cells during angiogenesis (Klein-Soyer et al., 2000, Arterioscler. Thromb. Vasc. Biol., 20: 360-9). The overexpression of CD9 in adenocarcinoma cells suppresses their motility and metastatic potential (Ikeyama et al., 1993, J. Exp. Med., 177: 1231-1237); its expression is inversely correlated with the primary tumors and the appearance of metastases in melanomas, lung cancer, colon cancer and breast cancer (Si and Hersey, 1993, Int. J. Cancer, 54, 37-43; Miyake et al., 1995, Cancer Res., 55: 4127-4131; Adachi et al., 1998, J. Clin. Oncol., 15, 1397-1406; Mori et al., 1998, Clin. Cancer Res., 4, 1507-1510).
The protein CD9P-1 (or FPLP) was identified as being the major partner molecule of CD9 in cancer lines (Serru et al., 1999, Biochem. J., 340, 103-111).
It has also been reported that the protein CD81 is implicated in various functions such as cell signalization and activation of the B lymphocytes (Fearon and Carter, 1995), regulation of the proliferation of the T lymphocytes (Miyazaki et al., 1997, EMBO J., 16, 4217-4225); it could also play a role in cancer because CD81 is a possible receptor for hepatitis C virus, a major cause of hepatic carcinoma (Pileri et al., 1998, Science, 282, 938-941).
Although the exact role of CD9P-1 or FPRP has yet to be defined, its association with CD9 or CD81 can suggest a role in the regulation functions of the CD9 or CD81 receptors. However, no role in the regulation of angiogenesis has been reported to date for the protein called angiopartnerine, identified by the number SEQ ID No. 6 in attached sequence listing, nor for the proteins PTGFRN, CD9P-1/FPRP.
The protein NKX3.1 is a member of the NK class of Homeobox proteins, closely linked to the protein NK-3 of Drosophila (Kim, Y. and Nirenberg, 1989, Proc. Natl. Acad. Sci. USA, 86, 7716-7720; He et al., 1997, Genomics, 43, 69-77). Studies on the mouse showed the expression of the gene NKX3.1 in the fetus and embryo in development in a variety of tissue types such as the mesoderm, vascular smooth muscle, epithelium and regions of the central nervous system (Kos et al., 1998, Mech. Dev., 70, 25-34; Tanaka et al., 1999, Mech. Dev., 85, 179-182; Bhatia-Gaur et al., 1999, Genes Dev., 13, 966-977). In the adult, the protein NKX3.1 is localized predominantly in the prostate, more particularly in the epithelial cells, and its expression is regulated by the androgens (He et al., 1997, Genomics, 43, 69-77; Prescott et al., 1998, Prostate, 35, 71-80; Sciavolino et al., 1997, Dev. Dyn., 209, 127-138; Ornstein et al., 2001, J. Urol., 165(4): 1329-34). It appears to play an essential role in the function of the prostate and regulates the proliferation of the epithelial cells of the prostate; the gene NKX3.1 was proposed to be a suppressor gene of the specific tumors of the prostate (Bhatia-Gaur et al., 1999, Genes Dev., 13(8): 966-966). The loss of expression of NKX3.1 in the human cancers of the prostate was recently correlated with the progression of tumors (Bowen et al., 2000, Cancer Res., 60(21): 6111-5). Moreover, it has already been reported that the homeobox proteins are implicated in the regulation of angiogenesis (review: Gorski and Walsh, 2000, Circulation Research, 87: 865-872).
However, no role for the homeobox protein NKX3.1 has been reported to date in the regulation of angiogenesis.
The protein hZFH (human zinc-finger helicase) belongs to the family of Snf2 type helicases known to act as transcriptional regulators for multiple genes (Aubry et al., 1998, Eur. J. Biochem., 243(3): 558-64). It also contains a chromodomain and is homologous with the protein CHD3 (“chromodomain helicase DNA binding protein 3”) identified by the sequence SEQ ID No. 29 and SEQ ID No. 30 in the attached sequence listing. It has been reported that the CHD3 proteins could regulate the expression of genes by repressing transcription via an alteration of the structure of chromatin (Zhang et al., 1998, Cell, 95, 279-289; Kehle et al., 1998, Science, 282, 1898-1900). No role has been reported to date in the regulation of the expression of the genes implicated in angiogenesis either for the protein hZFH or for the protein CHD3.
Factor 3 of initiation of eukaryote translation (EIF3), the largest initiation factor of protein synthesis, with a size of 650 kDa, is composed of at least nine peptide subunits (Hershey et al., 1996, Biochimie, 78, 903-907), including subunit 8 (p110) (Asano et al., 1997, J. Biol. Chem., 272, 1101-1109). EIF3 plays a central role in the initiation process of protein biosynthesis notably in the binding of the initiator methionyl-tRNA and mRNA to the ribosome subunit 40S so as to form the initiation complex 40S (Merrick and Hershey, 1996, The pathway and mechanisms of eukaryotic protein synthesis. In: Hershey J W B, Mathews M B, Sonenberg N, eds. Translational Control. Cold Spring Harbor, N.Y.: Cold Spring Harbor Press; 1996; 31-67). EIF3 appears to play a central role in the initiation by interaction with numerous other translational components (Vornlocher et al., 1999, J. Biol. Chem., Vol. 274, Issue 24, 16802-16812). The functions of each subunit are still poorly understood. High levels of expression of certain subunits are detected in tumors such as p150, p170; it has been proposed that p170 plays another role in addition to its functions in the initiation of translation (Lin et al., 2001, J. Cell Biochem., 80(4): 483-90; Pincheira et al., 2000, Eur. J. Cell Biol., 80(6): 410-8). Overexpression of subunit 8 (p 110) has also been demonstrated in a tumor of the germinal cells by Roche et al. (2000, American Journal of Pathology, 157: 1597-1604) which suggests a role of this subunit in the development of the tumor by augmenting translation in general, leading to augmented growth and cell division.
No implication in the regulation of angiogenesis of subunit 8 of EIF3 nor of its similar protein have been reported to date.
The control of angiogenesis thus represents a strategic axis both for fundamental research (in order to improve the comprehension of numerous pathological phenomena linked to angiogenesis) and for the development of new therapies intended to treat pathologies linked to angiogenesis.
In order to control angiogenesis, multiple pharmaceutical groups have therefore developed therapeutic strategies based directly on the use of paracrine stimulatory and inhibitory factors as agents for promoting or inhibiting angiogenesis. These strategies are based essentially on the use of such factors in their polypeptide form as stimulatory or inhibitory agents of angiogenesis, or more recently in the form of expression vectors coding for the selected factors.